Cyclic nucleotide phosphodiesterases show specificity for purine cyclic nucleotide substrates and catalyze cyclic AMP (cAMP) and cyclic GMP (cGMP) hydrolysis (Thompson W. J. (1991) Pharma. Ther. 51:13-33). cyclic nucleotide phosphodiesterases regulate the steady-state levels of cAMP and cGMP and modulate both the amplitude and duration of cyclic nucleotide signal. At least eight different but homologous gene families are currently known to exist in mammalian tissues. Most families contain distinct genes, many of which are expressed in different tissues as functionally unique alternative splice variants. (Beavo (1995) Physiological Reviews 75:725-748 and U.S. Pat. No. 5,798,246).
All cyclic nucleotide phosphodiesterases contain a core of about 270 conserved amino acids in the COOH-terminal half of the protein thought to be the catalytic domain of the enzyme. A conserved motif of the sequence HDXXHXX (SEQ ID NO:40 ) has been identified in the catalytic domain of all cyclic nucleotide phosphodiesterases isolated to date. The cyclic nucleotide phosphodiesterases within each family display about 65% amino acid homology and the similarity drops to less than 40% when compared between different families with most of the similarity occurring in the catalytic domains.
Most cyclic nucleotide phosphodiesterase genes have more than one alternatively spliced mRNA transcribed from them and in many cases the alternative splicing appears to be highly tissue specific, providing a mechanism for selective expression of different cyclic nucleotide phosphodiesterases (Beavo supra). Cell-type-specific expression suggests that the different isozymes are likely to have different cell-type-specific properties.
Type 1 cyclic nucleotide phosphodiesterases are Ca.sup.2+ /calmodulin dependent, are reported to contain three different genes, each of which appears to have at least two different splice variants, and have been found in the lung, heart and brain. Some of the calmodulin-dependent phosphodiesterases are regulated in vitro by phosphorylation/dephosphorylation events. The effect of phosphorylation is to decrease the affinity of the enzyme for calmodulin, which decreases phosphodiesterase activity, thereby increasing the steady state level of cAMP. Type 2 cyclic nucleotide phosphodiesterases are cGMP stimulated, are localized in the brain and are thought to mediate the effects of cAMP on catecholamine secretion. Type 3 cyclic nucleotide phosphodiesterases are cGMP-inhibited, have a high specificity for cAMP as a substrate, and are one of the major phosphodiesterase isozymes present in vascular smooth muscle and play a role in cardiac function. One isozyme of type 3 is regulated by one or more insulin-dependent kinases. Type 4 cyclic nucleotide phosphodiesterases are the predominant isoenzyme in most inflammatory cells, with some of the members being activated by cAMP-dependent phosphorylation. Type 5 cyclic nucleotide phosphodiesterases have traditionally been thought of as regulators of cGMP function but may also affect cAMP function. High levels of type 5 cyclic nucleotide phosphodiesterases are found in most smooth muscle preparations, platelets and kidney. Type 6 cyclic nucleotide phosphodiesterase family members play a role in vision and are regulated by light and cGMP. A Type 7 cyclic nucleotide phosphodiesterase family member is found in high concentrations in skeletal muscle. A listing of cyclic nucleotide phosphodiesterase families 1-7, their localization and physiological role is given in Beavo supra. A Type 8 family is reported in U.S. Pat. No. 5,798,246.
Many functions of the immune and inflammatory responses are inhibited by agents that increase intracellular levels of cAMP (Verghese (1995) Mol. Pharmacol. 47:1164-1171) while the metabolism of cGMP is involved in smooth muscle, lung and brain cell function (Thompson W. (1991) Pharma. Ther. 51:13-33). A variety of diseases have been attributed to increased cyclic nucleotide phosphodiesterase activity which results in decreased levels of cyclic nucleotides. For example, one form of diabetes insipidus in the mouse has been associated with increased phosphodiesterase Family 4 activity and an increase in low-Km cAMP phosphodiesterase activity has been reported in leukocytes of atopic patients. Defects in cyclic nucleotide phosphodiesterases have also been associated with retinal disease. Retinal degeneration in the rd mouse, human autosomal recessive retinitis pigmentosa, and rod/cone dysplasia 1 in Irish setter dogs has been attributed to mutations in the Family 6 phosphodiesterase, gene B. Family 3 phosphodiesterase has been associated with cardiac disease.
Many inhibitors of different cyclic nucleotide phosphodiesterases have been identified and some have undergone clinical evaluation. For example, Family 3 phosphodiesterase inhibitors are being developed as antithrombotic agents, as antihypertensive agents and as cardiotonic agents useful in the treatment of congestive heart failure. Rolipram, a Family 4 phosphodiesterase inhibitor, has been used in the treatment of depression and other inhibitors of Family 4 phosphodiesterase are undergoing evaluation as anti-inflammatory agents. Rolipram has also been shown to inhibit lipopolysaccharide (LPS) induced TNF-alpha which has been shown to enhance HIV-1 replication in vitro. Therefore, rolipram may inhibit HIV-1 replication (Angel et al. (1995) AIDS 9:1137-44). Additionally, based on its ability to suppress the production of TNF alpha and beta and interferon gamma, rolipram has been shown to be effective in the treatment of encephalomyelitis, the experimental animal model for multiple sclerosis (Sommer et al. (1995) Nat. Med. 1:244-248) and may be effective in the treatment of tardive dyskinesia (Sasaki et al. (1995) Eur. J. Pharmacol. 282:72-76).
There are also nonspecific phosphodiesterase inhibitors such as theophylline, used in the treatment of bronchial asthma and other respiratory diseases, and pentoxifylline, used in the treatment of intermittent claudication and diabetes-induced peripheral vascular disease. Theophylline is thought to act on airway smooth muscle function as well as in an anti-inflammatory or immunomodulatory capacity in the treatment of respiratory diseases (Banner et al. (1995) Eur. Respir. J 8:996-1000) where it is thought to act by inhibiting both cyclic nucleotide phosphodiesterase cAMP and cGMP hydrolysis (Banner et al. (1995) Monaldi Arch. Chest Dis. 50:286-292). Pentoxifylline, also known to block TNF-alpha production, may inhibit HIV-1 replication (Angel et al. supra). A list of cyclic nucleotide phosphodiesterase inhibitors is given in Beavo supra.
Cyclic nucleotide phosphodiesterases have also been reported to affect cellular proliferation of a variety of cell types and have been implicated in the treatment of various cancers. (Bang et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334) reported that the prostate carcinoma cell lines DU 145 and LNCaP were growth-inhibited by delivery of cAMP derivatives and phosphodiesterase inhibitors and observed a permanent conversion in phenotype from epithelial to neuronal morphology; Matousovic et al. ((1995) J. Clin. Invest. 96:401-410) suggest that cyclic nucleotide phosphodiesterase isozyme inhibitors have the potential to regulate mesangial cell proliferation; Joulain et al. ((1995) J. Mediat. Cell Signal 11:63-79) reports that cyclic nucleotide phosphodiesterase has been shown to be an important target involved in the control of lymphocyte proliferation; and Deonarain et al. ((1994) Brit. J. Cancer 70:786-94) suggest a tumor targeting approach to cancer treatment that involves intracellular delivery of phosphodiesterases to particular cellular compartments, resulting in cell death.
Accordingly, cyclic nucleotide phosphodiesterases are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown phosphodiesterases. The present invention advances the state of the art by providing a previously unidentified human cyclic nucleotide phosphodiesterase.